Project summary Bone mass is determined by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. Osteocytes, post-mitotic cells embedded within bone, control this balance by producing paracrine factors that regulate osteoblast and osteoclast activity. Therefore, therapies that target osteocytes represent promising new strategies to treat post-menopausal osteoporosis. Osteocytes respond to external cues, such as parathyroid hormone, to orchestrate bone remodeling. A crucial step in the signaling cascade through which osteocytes respond to PTH is inhibition of the kinase salt inducible kinase 2 (SIK2). Small molecule SIK2 inhibitors, such as YKL-05-099, mimic PTH action. Despite these advances, major knowledge gaps currently exist in our understanding of how the PTH/SIK signaling axis regulates skeletal biology. Aim 1 of this proposal will determine the role of salt inducible kinases in osteocytes in vivo. Mice lacking both SIK2 and SIK3 in DMP1-Cre-expressing cells have been generated, and display skeletal phenotypes quite reminiscent of hyperparathyroidism. Detailed bone phenotypic analysis of these mice will be performed, focusing on similarities between hyperparathyroid bone disease. Of the SIK isoforms expressed in osteocytes, SIK2 is uniquely PTH-responsive, and acts as a switch responsible for PTH-mediated substrate dephosphorylation and target gene expression. SIK2 conditional knockout mice will be treated with intermittent PTH once daily for 4 weeks, and resultant bone phenotypes assessed by advanced radiographic and histologic techniques. Aim 2 of this proposal will define the contribution of distinct SIK substrates in PTH responses in osteocytes. We currently know that PTH-mediated HDAC4/5 dephosphorylation regulates sclerostin expression, and that PTH- mediated CRTC2 dephosphorylation controls RANKL upregulation. However, PTH regulates a large number of target genes in osteocytes, and the relative contributions of these events to the overall response to parathyroid hormone remains unknown. Furthermore, whether PTH regulates phosphorylation of additional SIK substrates is not known. We have performed phosphoproteomic profiling to identify novel phosphoproteins whose abundance is decreased by both PTH and small molecule SIK inhibitors. In doing so, we identified FOXO3 is a novel SIK substrate. Here, the mechanisms through which PTH regulates FOXO3 phosphorylation, subcellular localization, and target gene expression will be explored. In addition, we will employ loss of function approaches to study the relative contribution of CRTC2 and FOXO3 in PTH-regulated target gene expression in vitro and in vivo. Taken together, these studies will significantly advance our knowledge of how the PTH/SIK signaling axis controls osteocyte biology. A detailed understanding of the steps linking SIK inhibition and regulation of gene expression will illuminate novel mechanisms through which parathyroid hormone acts in bone. Furthermore, this work will identify novel osteoporosis drug targets, and significantly facilitate the development of SIK inhibitors as bone anabolic treatment agents.